Linoleic Acid Induces Metabolic Reprogramming and Inhibits Oxidative and Inflammatory Effects in Keratinocytes Exposed to UVB Radiation.

Linoleic acid (LA), the primary ω-6 polyunsaturated fatty acid (PUFA) found in the epidermis, plays a crucial role in preserving the integrity of the skin's water permeability barrier. Additionally, vegetable oils rich in LA have been shown to notably mitigate ultraviolet (UV) radiation-induced effects, including the production of reactive oxygen species (ROS), cellular damage, and skin photoaging. These beneficial effects are primarily ascribed to the LA in these oils. Nonetheless, the precise mechanisms through which LA confers protection against damage induced by exposure to UVB radiation remain unclear. This study aimed to examine whether LA can restore redox and metabolic equilibria and to assess its influence on the inflammatory response triggered by UVB radiation in keratinocytes. Flow cytometry analysis unveiled the capacity of LA to diminish UVB-induced ROS levels in HaCaT cells. GC/MS-based metabolomics highlighted significant metabolic changes, especially in carbohydrate, amino acid, and glutathione (GSH) metabolism, with LA restoring depleted GSH levels post-UVB exposure. LA also upregulated PI3K/Akt-dependent GCLC and GSS expression while downregulating COX-2 expression. These results suggest that LA induces metabolic reprogramming, protecting against UVB-induced oxidative damage by enhancing GSH biosynthesis via PI3K/Akt signaling. Moreover, it suppresses UVB-induced COX-2 expression in HaCaT cells, making LA treatment a promising strategy against UVB-induced oxidative and inflammatory damage.

Financial impact of low pathogenic avian influenza virus subtype H9N2 on commercial broiler chicken and egg layer production systems in Pakistan.

Low Pathogenic Avian Influenza (LPAI) subtype H9N2 is endemic in Pakistan and impacts poultry farming through disease related mortality, poor weight gain and reduced egg production. This study aims to estimate the farm-level financial impact of LPAI H9N2 infection on commercial broiler and layer production systems in Pakistan. A questionnaire based cross-sectional survey of 138 broiler farms and 136 layer farms in Pakistan was conducted in 2019. Primary data collected by cross-sectional survey along with expert opinion and published literature were used to parameterize five stochastic production and gross margin models for three broiler and two layer production systems: fully integrated production (FIP), partially integrated production (PIP) and independent farming production (IP) systems. Partial budget analysis were then carried out to estimate the financial impact of LPAI H9N2. Results indicate that in broiler production systems, starting with 35,000 day old chicks (DOC) per batch, the net cost of disease (million PKR/production cycle) was estimated at 4.10 (14,862 USD), 4.62 (16,747 USD) and 2.46 (8917 USD) for IP, PIP and FIP systems, respectively. The disease produced a negative gross margin (defined here as revenue minus replacement and variable costs) in IP (-53 PKR (-0.19 USD)/DOC bought) and PI (-25 PKR (-0.091 USD)/DOC bought) systems, while remained positive for FIP systems (87 PKR (0.32 USD)/DOC bought). For layer production systems, (mean flock size as 48,000 DOCs) the net cost (million PKR/production cycle) was 29.75 (107,095.21 USD) and 29.51 (106,223.45 USD) IP and PIP systems, respectively, and produced negative gross margin in both systems. The outcomes of the study highlight the vulnerability of independent and partially integrated production systems to the disease. These findings also offer a decision-making tool to the farmers and policy makers to evaluate avian influenza surveillance systems and control interventions in Pakistan.

Health infrastructure evaluations of trading partners for equivalence: Why they are important and current hurdles for One Health collaboration.

The history of trade has numerous examples of movement of diseases and pests. Nations institute barriers to protect human, animal, and plant populations from incursions; sometimes successfully, sometimes not. The World Trade Organization and international standard-setting bodies (ISSB) are multilateral organizations that promote equitable treatment and safe trade. Equivalence of outcome is a concept that crosses animal, human, and environmental health standards. Loosely defined, it is taken to mean achieving a similar reduction in risk by different methods. Countries conduct assessments known as health infrastructure evaluations (HIE) to allow safe trade in commodities of food, plant, and animal origin. This review analyzes the use of HIE for trade purposes, with an added focus on their One Health context. The goal is to identify areas of HIE in which a One Health approach could assist in achieving the goal of "treating like partners alike," thereby fostering transparent decision-making in trade negotiations, a key obligation under World Trade Organization rules.

Évaluations des infrastructures de santé des partenaires commerciaux pour l'équivalence : pourquoi elles sont importantes et des obstacles actuels à la collaboration Une seule santéL'histoire du commerce regorge d'exemples de mouvements de maladies et de ravageurs. Les nations instituent des barrières pour protéger les populations humaines, animales et végétales des incursions; parfois avec succès, parfois non. L'Organisation mondiale du commerce et les organismes internationaux de normalisation (ISSB) sont des organisations multilatérales qui font la promotion d'un traitement équitable et d'un commerce sûr. L'équivalence des résultats est un concept qui traverse les normes de santé animale, humaine et environnementale. De manière vague, cela signifie parvenir à une réduction similaire du risque par différentes méthodes. Les pays mènent des évaluations connues sous le nom d'évaluations des infrastructures sanitaires (HIE) pour permettre un commerce sûr des produits d'origine alimentaire, végétale et animale. Cette revue analyse l'utilisation des HIE à des fins commerciales, en mettant davantage l'accent sur leur contexte Une seule santé. L'objectif est d'identifier les domaines de l'HIE dans lesquels une approche Une seule santé pourrait contribuer à atteindre l'objectif de « traiter comme des partenaires sur un pied d'égalité ¼, favorisant ainsi une prise de décision transparente dans les négociations commerciales, une obligation clé en vertu des règles de l'Organisation mondiale du commerce.(Traduit par Dr Serge Messier).

Determinants of Thoroughbred yearling sales price in the UK.

Background: Industry-level figures suggest that up to two-thirds of Thoroughbred breeding operations in the UK are unprofitable and that around half of sales transactions of Thoroughbred yearlings, commercial breeders' predominant income source, return a loss. The industry strategy currently endorses investment in stallion covering fee; however, to date, a comprehensive evaluation of sales price determinants in the UK setting is lacking and could better inform economic decision making to improve profitability. Methods: Sales catalogue and Weatherbys' stud book data from all Thoroughbred yearlings sold at the 2020 Tattersalls October yearling sale in the UK were used to build a hedonic sales price model. Explanatory variables representing sire, dam, yearling and sales attributes were evaluated. The final model's accuracy was assessed using out-of-sample data from all yearlings sold in the equivalent 2021 sale. Results: In 2020, a total of 1506 catalogued yearlings, representing around 30% of the UK Thoroughbred foal crop, were sold, with a median price of £42,575 (interquartile range 15,750‒105,000; range 840‒3,570,000). The sires' covering fee, maternal siblings' race performance attributes, whether the yearling was the dams' first foal, consignment size, catalogue book and day of sale within book significantly influenced auction price; however, relationships were complex with significant interaction and confounding observed. The mean model forecasting error was £2074. The use of data from only one sale could affect generalisability. Conclusions: These novel findings can inform breeding decisions to maximise profitability, give context for current industry strategies and can inform valuations of breeding stock.

A Genetic Tool to Identify Predators Responsible for Livestock Attacks in South America and Recommendations for Human-Wildlife Conflict Mitigation.

Livestock predation induces global human-wildlife conflict, triggering the retaliatory killing of large carnivores. Although domestic dogs (Canis familiaris) contribute to livestock depredation, blame primarily falls on wild predators. Dogs can also transmit pathogens between wildlife, domestic animals, and humans. Therefore, the presence of free-ranging dogs can have negative consequences for biodiversity conservation, smallholder economy, food supply, and public health, four of the United Nations' Sustainable Developed Goals (SDGs) for 2030. In Ecuador, where livestock sustains rural households, retaliatory poaching threatens Andean bear (Tremarctos ornatus), jaguar (Panthera onca), and puma (Puma concolor) populations. However, the role of dogs in these incidents remains underexplored. The present study evaluates the possibility of reliable molecular identification of predatory species from DNA traces in bite wounds. Our results revealed the presence of dog saliva on four out of six livestock carcasses presumably attacked by wild predators. These findings highlight the importance of rectifying misinformation about large carnivores in Ecuador and the need to control dog populations. We recommend that local administrations incorporate DNA analysis into livestock predation events to examine how common the problem is, and to use the analysis to develop conflict mitigation strategies which are essential for the conservation of large carnivores.

Andrographolide Ameliorates Inflammatory Changes Induced by D-Lactate in Bovine Fibroblast-like Synoviocytes.

During acute ruminal acidosis, the manifestation of aseptic polysynovitis and lameness in cattle has been observed. Evidence suggests that joint inflammation can be attributed to the metabolic alterations induced by D-lactate in fibroblast-like synoviocytes (FLSs). We aimed to investigate whether andrographolide could mitigate the inflammation and metabolic alterations induced by D-lactate in bovine fibroblast-like synoviocytes (bFLSs). To assess this, bFLSs were cultured in the presence or absence of andrographolide. We evaluated its potential interference with the expression of proinflammatory cytokines, COX-2, HIF-1α, and LDHA using RT-qPCR. Furthermore, we investigated its potential interference with PI3K/Akt signaling and IκBα degradation through immunoblotting and flow cytometry, respectively. Our observations revealed that andrographolide reduced the elevation of IL-6, IL-8, COX-2, HIF-1α, and LDHA induced by D-lactate. Additionally, andrographolide demonstrated interference with the PI3K/Akt and NF-κB pathways in bFLSs. In conclusion, our findings suggest that andrographolide can potentially reverse the inflammatory effects and metabolic changes induced by D-lactate in bFLSs, showing promise as a therapeutic intervention for managing these conditions associated with lameness.

Neural tube defects and epigenetics: role of histone post-translational histone modifications.

Neural tube defects (NTDs) are the most common congenital anomalies of the CNS. It is widely appreciated that both genetic and environmental factors contribute to their etiology. The inability to ascribe clear genetic patterns of inheritance to various NTD phenotypes suggests it is possible that epigenetic mechanisms are involved in the etiology of NTDs. In this context, the contribution of DNA methylation as an underlying contributing factor to the etiology of NTDs has been extensively reviewed. Here, an updated accounting of the evidence linking post-translational histone modifications to these birth defects, relying heavily upon studies in humans, and the possible molecular implications inferred from reports based on cellular and animal models, are presented.

Thermoneutral Housing Enables Studies of Vertical Transmission of Obesogenic Diet-Driven Metabolic Diseases.

Vertical transmission of obesity is a critical contributor to the unabated obesity pandemic and the associated surge in metabolic diseases. Existing experimental models insufficiently recapitulate "human-like" obesity phenotypes, limiting the discovery of how severe obesity in pregnancy instructs vertical transmission of obesity. Here, via utility of thermoneutral housing and obesogenic diet feeding coupled to syngeneic mating of WT obese female and lean male mice on a C57BL/6 background, we present a tractable, more "human-like" approach to specifically investigate how maternal obesity contributes to offspring health. Using this model, we found that maternal obesity decreased neonatal survival, increased offspring adiposity, and accelerated offspring predisposition to obesity and metabolic disease. We also show that severe maternal obesity was sufficient to skew offspring microbiome and create a proinflammatory gestational environment that correlated with inflammatory changes in the offspring in utero and adulthood. Analysis of a human birth cohort study of mothers with and without obesity and their infants was consistent with mouse study findings of maternal inflammation and offspring weight gain propensity. Together, our results show that dietary induction of obesity in female mice coupled to thermoneutral housing can be used for future mechanistic interrogations of obesity and metabolic disease in pregnancy and vertical transmission of pathogenic traits.

Assessing antibiotic usage data capture accuracy on dairy farms in England and Wales.

BACKGROUND: Accurate farm-level data on antibiotic usage (ABU) are needed for the surveillance of antibiotic resistance. Therefore, this study aimed to determine the accuracy of ABU data capture by dairy farmers in South West England and Wales. METHODS: Through a cross-sectional survey of 48 dairy farmers, the accuracy of ABU recording was measured by farmers' assessment of the completeness and timeliness of ABU recording ('perceived accuracy') and the completeness and correctness of on-farm ABU records ('actual accuracy'). Completeness and correctness were compared for paper and software recording methods. RESULTS: Perceived accuracy was higher than actual accuracy. Antibiotic names, withdrawal periods and dates that products were fit for human consumption were often incomplete or incorrect. More inaccuracies were seen with paper than software. In some software platforms, the date that milk would be fit for human consumption was frequently rounded down by half a day, increasing the risk of residue failures. LIMITATION: The small number of on-farm records assessed limits the generalisability of the results. CONCLUSIONS: Electronic recording of ABU should be encouraged. However, functionality needs improvement, alongside consultation with dairy farmers to increase awareness of inaccuracies.

Obesity amplifies influenza virus-driven disease severity in male and female mice.

Influenza virus-induced respiratory pneumonia remains a major public health concern. Obesity, metabolic diseases, and female sex are viewed as independent risk factors for worsened influenza virus-induced lung disease severity. However, lack of experimental models of severe obesity in female mice limits discovery-based studies. Here, via utility of thermoneutral housing (30 °C) and high-fat diet (HFD) feeding, we induced severe obesity and metabolic disease in female C57BL/6 mice and compared their responses to severely obese male C57BL/6 counterparts during influenza virus infection. We show that lean male and female mice have similar lung edema, inflammation, and immune cell infiltration during influenza virus infection. At standard housing conditions, HFD-fed male, but not female, mice exhibit severe obesity, metabolic disease, and exacerbated influenza disease severity. However, combining thermoneutral housing and HFD feeding in female mice induces severe obesity and metabolic disease, which is sufficient to amplify influenza virus-driven disease severity to a level comparable to severely obese male counterparts. Lastly, increased total body weights of male and female mice at time of infection correlated with worsened influenza virus-driven disease severity metrics. Together, our findings confirm the impact of obesity and metabolic disease as key risk factors to influenza disease severity and present a novel mouse experimental model suitable for future mechanistic interrogation of sex, obesity, and metabolic disease traits in influenza virus-driven disease severity.

Understanding household and food system determinants of chicken and egg consumption in India.

Poultry is one of the fastest-growing agricultural sectors in India and its demand is said to be rising. There is a perception that higher incomes, growing population, urbanisation, and increased productivity in the industry have influenced Indian poultry consumption. However, consumer surveys have shown that the average poultry consumption in India has remained low. With this in mind, the paper analysed household determinants of chicken and egg consumption within the Indian population, using two rounds of National Sample Survey data (1993-1994 and 2011-2012). By conducting a spatiotemporal analysis of household consumption and expenditure survey and by using truncated Double Hurdle and Unconditional Quantile regressions (UQR) models, this study explored socio-economic and food system determinants of chicken and egg consumption in India. Key results highlight that while consumption has increased marginally over twenty years, supply-side determinants, such as price and poultry production concentration, influenced heterogenous consumption patterns in India. We also find evidence that historically marginalised groups consumed more chicken and eggs in comparison to non-marginalised groups and preliminary evidence suggests how household gender dynamics influence different consumption patterns. Adequate consumption of poultry is important to improve nutrient-deficient diets of vulnerable groups in India. Our findings on demand side determinants of poultry products are crucial to support consumer tailored actions to improve nutritional outcomes along with the Indian poultry sector policy planning.

d-lactate-induced ETosis in cattle polymorphonuclear leucocytes is dependent on the release of mitochondrial reactive oxygen species and the PI3K/Akt/HIF-1 and GSK-3ß pathways.

d-lactate is a metabolite originating from bacterial metabolism that accumulates as a result of dietary disturbances in cattle, leading to ruminal acidosis. d-lactate exerts functions as a metabolic signal inducing metabolic reprogramming and extracellular trap (ET) release in polymorphonuclear leucocytes (PMNs). We previously demonstrated that d-lactate induces metabolic reprogramming via hypoxia-induced factor 1 alpha (HIF-1α) stabilization in bovine fibroblast-like synoviocytes (FLSs). In the present study, the role of HIF-1 in ET formation induced by d-lactate was assessed. HIF-1α stabilization in PMNs was controlled by mitochondrial reactive oxygen species (mtROS) release. Furthermore, inhibition of mitochondrial complex I and scavenging of mtROS decreased d-lactate-triggered ETosis. d-lactate-enhanced HIF-1α accumulation was dependent on the PI3K/Akt pathway but independent of GSK-3ß activity. Pharmacological blockade of the PI3K/Akt/HIF-1 and GSK-3ß axes inhibited d-lactate-triggered ETosis and downregulated PDK1 and LDHA expression. However, only GSK-3ß inhibition decreased the expression of glycogen metabolism enzymes and prevented the decline in glycogen stores induced by d-lactate exposure. The results of this study suggest that mtROS, PI3K/Akt/HIF-1 and GSK-3ß axes regulate carbohydrate metabolism adaptations that support d-lactate-induced ET formation in cattle.

Mapping chicken production and distribution networks in Vietnam: An analysis of socio-economic factors and their epidemiological significances.

The growing chicken industry in Viet Nam has an increasingly important contribution to the country's food security, but its development requires careful planning to prevent disease risks. This study characterizes the chicken production and distribution networks in Vietnam and identifies potential factors that could promote disease emergence and transmission. Qualitative data were collected from interviews with 29 key informants from five stakeholder groups representing the main nodes from chicken production and distribution networks (PDN). Three main networks were identified based on production type: a colored broiler and spent hen network, a white (or exotic) broiler network, and an egg network. Colored chickens and spent hens are the most preferred commodity by vietnamese consumers and their PDN is composed of production units differing in their scale and management and with long distribution chains involving numerous small-scale independent stakeholders. Live bird markets plays a central role in this network, which is driven by consumers' preference for live chickens. The white chicken network presents an important duality, as it is composed of both a large number of independent household farms and traders operating independently with little chain coordination, and of large farms contracted by vertically-integrated companies. The egg PDN was the most organized network, being mostly controlled by large vertically-integrated companies. High level specialization and diversification of stakeholders is found in all three networks. Stakeholders' perceptions of the main factors promoting disease risk along the PDN were the low biosecurity in household farms and live bird markets, mobile traders, the informal slaughter of birds and the management of sick birds. Findings from this study can be used to plan future studies to support food system planners in the development of safer poultry production and distribution in Vietnam.

ATP Induces Interleukin-8, Intracellular Calcium Release, and ERK1/2 Phosphorylation in Bovine Endometrial Cells, Partially through P2Y Receptors.

The bovine endometrium has an important defensive role in the postpartum period that acts when an inflammatory process associated with tissue damage or infection by bacteria is produced. Endometrial cells release cytokines and chemokines that recruit inflammatory cells, which release danger-associated molecular patterns (DAMPs), such as adenosine triphosphate (ATP), and initiate and regulate the inflammatory response. However, the role of ATP in bovine endometrial cells is unclear. The aim of this study was to determine the effect of ATP on interleukin-8 (IL-8) release, intracellular calcium mobilization, ERK1/2 phosphorylation, and the role of P2Y receptors, in bovine endometrial cells. Bovine endometrial (BEND) cells were incubated with ATP and the IL-8 release was determined by the ELISA assay. ATP of 50 and 100 µM significantly increased IL-8 released in BEND cells (50 µM: 23.16 ± 3.82 pg/mL, p = 0.0018; 100 µM: 30.14 ± 7.43 pg/mL, p = 0.0004). ATP (50 µM) also induced rapid intracellular calcium mobilization in Fura-2AM-loaded BEND cells, as well as ERK1/2 phosphorylation (ratio 1.1 ± 0.04, p = 0.0049). Suramin (50 µM), a pan-antagonist of P2Y receptors, partially reduced the intracellular calcium mobilization, ERK1/2 phosphorylation (ratio 0.83 ± 0.08, p = 0.045), and IL-8 release (9.67 ± 0.02 pg/mL, p = 0.014) induced by ATP. Finally, BEND cells expressed higher mRNA levels of P2Y1 and P2Y2 purinergic subtype receptors, and lower levels of P2Y11 and P2Y12 receptors, as determined by RT-qPCR. In conclusion, these results showed that ATP activates pro-inflammatory responses in BEND cells, which are partially mediated via P2Y receptors, and BEND cells express the mRNA of subtypes of P2Y receptors, which could have a key role in bovine endometrial inflammation.

Thermoneutral housing shapes hepatic inflammation and damage in mouse models of non-alcoholic fatty liver disease.

Introduction: Inflammation is a common unifying factor in experimental models of non-alcoholic fatty liver disease (NAFLD) progression. Recent evidence suggests that housing temperature-driven alterations in hepatic inflammation correlate with exacerbated hepatic steatosis, development of hepatic fibrosis, and hepatocellular damage in a model of high fat diet-driven NAFLD. However, the congruency of these findings across other, frequently employed, experimental mouse models of NAFLD has not been studied. Methods: Here, we examine the impact of housing temperature on steatosis, hepatocellular damage, hepatic inflammation, and fibrosis in NASH diet, methionine and choline deficient diet, and western diet + carbon tetrachloride experimental models of NAFLD in C57BL/6 mice. Results: We show that differences relevant to NAFLD pathology uncovered by thermoneutral housing include: (i) augmented NASH diet-driven hepatic immune cell accrual, exacerbated serum alanine transaminase levels and increased liver tissue damage as determined by NAFLD activity score; (ii) augmented methionine choline deficient diet-driven hepatic immune cell accrual and increased liver tissue damage as indicated by amplified hepatocellular ballooning, lobular inflammation, fibrosis and overall NAFLD activity score; and (iii) dampened western diet + carbon tetrachloride driven hepatic immune cell accrual and serum alanine aminotransferase levels but similar NAFLD activity score. Discussion: Collectively, our findings demonstrate that thermoneutral housing has broad but divergent effects on hepatic immune cell inflammation and hepatocellular damage across existing experimental NAFLD models in mice. These insights may serve as a foundation for future mechanistic interrogations focused on immune cell function in shaping NAFLD progression.

Pharma-cartography: Navigating the complexities of antibiotic supply to rural livestock in West Bengal, India, through value chain and power dynamic analysis.

Antibiotic resistance threatens provision of healthcare and livestock production worldwide with predicted negative socioeconomic impact. Antibiotic stewardship can be considered of importance to people living in rural communities, many of which depend on agriculture as a source of food and income and rely on antibiotics to control infectious diseases in livestock. Consequently, there is a need for clarity of the structure of antibiotic value chains to understand the complexity of antibiotic production and distribution in community settings as this will facilitate the development of effective policies and interventions. We used a value chain approach to investigate how relationships, behaviours, and influences are established during antibiotic distribution. Interviews were conducted with key informants (n = 17), value chain stakeholders (n = 22), and livestock keeping households (n = 36) in Kolkata, and two rural sites in West Bengal, India. Value chain mapping and an assessment of power dynamics, using manifest content analysis, were conducted to investigate antibiotic distribution and identify entry points for antibiotic stewardship. The flow of antibiotics from manufacturer to stockists is described and mapped and two local level maps showing distribution to final consumers presented. The maps illustrate that antibiotic distribution occurred through numerous formal and informal routes, many of which circumvent antibiotic use legislation. This was partly due to limited institutional power of the public sector to govern value chain activities. A 'veterinary service lacuna' existed resulting in livestock keepers having higher reliance on private and informal providers, who often lacked legal mandates to prescribe and dispense antibiotics. The illegitimacy of many antibiotic prescribers blocked access to formal training who instead relied on mimicking the behaviour of more experienced prescribers-who also lacked access to stewardship guidelines. We argue that limited institutional power to enforce existing antibiotic legislation and guide antibiotic usage and major gaps in livestock healthcare services make attempts to curb informal prescribing unsustainable. Alternative options could include addressing public sector deficits, with respect to both healthcare services and antibiotic provision, and by providing resources such as locally relevant antibiotic guidelines to all antibiotic prescribers. In addition, legitimacy of informal prescribers could be revised, which may allow formation of associations or groups to incentivise good antibiotic practices.

Bovine tumor necrosis factor-alpha Increases IL-6, IL-8, and PGE2 in bovine fibroblast-like synoviocytes by metabolic reprogramming.

Lameness is a common condition in dairy cattle caused by infectious or noninfectious agents. Joint lesions are the second most common cause of lameness and can be diagnosed in association with the presentation of digit injuries. Fibroblast-like synoviocyte (FLS) are predominant cells of synovia and play a key role in the pathophysiology of joint diseases, thus increasing the expression of proinflammatory mediators. Tumor necrosis factor-alpha (TNF-α) is a potent proinflammatory cytokine involved in cyclooxygenase 2 (COX-2) and proinflammatory cytokine expression in FLS. Previously, TNF-α was demonstrated to increase hypoxia-inducible Factor 1 (HIF-1), a transcription factor that rewires cellular metabolism and increases the expression of interleukin (IL)-6 in bovine FLS (bFLS). Despite this, the proinflammatory effects of TNF-α in bFLS on metabolic reprogramming have been poorly studied. We hypothesized that TNF-α increases glycolysis and in this way controls the expression of IL-6, IL-8, and COX-2 in bFLS. Results first, gas chromatography/mass spectrometry (GC/MS)-based untargeted metabolomics revealed that bTNF-α altered the metabolism of bFLS, increasing glucose, isoleucine, leucine, methionine, valine, tyrosine, and lysine and decreasing malate, fumarate, α-ketoglutarate, stearate, palmitate, laurate, aspartate, and alanine. In addition, metabolic flux analysis using D-glucose-13C6 demonstrated an increase of pyruvate and a reduction in malate and citrate levels, suggesting a decreased flux toward the tricarboxylic acid cycle after bTNF-α stimulation. However, bTNF-α increased lactate dehydrogenase subunit A (LDHA), IL-6, IL-8, IL-1ß and COX-2 expression, which was dependent on glycolysis and the PI3K/Akt pathway. The use of FX11 and dichloroacetate (DCA), an inhibitor of LDHA and pyruvate dehydrogenase kinase (PDK) respectively, partially reduced the expression of IL-6. Our results suggest that bTNF-α induces metabolic reprogramming that favors glycolysis in bFLS and increases IL-6, IL-8, IL-1ß and COX-2/PGE2.

Hydroxycarboxylic acid receptor 2 (HCA2) agonists induce NET formation and MMP-9 release from bovine polymorphonuclear leukocytes.

Periparturient cows are commonly fed diets supplemented with Niacin (nicotinic acid, NA) because of its anti-lipolytic properties. NA confers its anti-lipolytic effects by activating the hydroxycarboxylic acid 2 receptor (HCA2). HCA2 is also activated by the ketone body beta-hydroxybutyrate (BHB) and circulating BHB levels are elevated in postpartum dairy cows. The HCA2 receptor is highly expressed in bovine polymorphonuclear leukocytes (PMN) and could link metabolic and innate immune responses in cattle. We investigated how HCA2 agonists affected bovine PMN function in vitro. We studied different PMN responses, such as granule release, surface expression of CD11b and CD47, generation of neutrophil extracellular traps (NETs), and apoptosis. NA, BHB, and 4,4aR,5,5aR-tetrahydro-1H-cyclopropa [4,5] cyclopenta [1,2-c] pyrazole-3-carboxylic acid (MK-1903) treatment triggered the release of matrix metalloproteinase 9 (MMP-9), a component of the tertiary granule, from neutrophils. Additionally, all HCA2 agonists induced NETs formation but did not affect surface expression of CD11b and CD47. Finally, none of the HCA2 agonists triggered apoptosis in bovine PMN. This information will give new insights into the potential role of the HCA2 receptor in the bovine innate immune response.

Glycolysis, monocarboxylate transport, and purinergic signaling are key events in Eimeria bovis-induced NETosis.

The protozoan parasite Eimeria bovis is the causative agent of bovine coccidiosis, an enteric disease of global importance that significantly affects cattle productivity. Previous studies showed that bovine NETosis-an important early host innate effector mechanism of polymorphonuclear neutrophil (PMN)-is elicited by E. bovis stages. So far, the metabolic requirements of E. bovis-triggered NET formation are unknown. We here studied early glycolytic and mitochondrial responses of PMN as well as the role of pH, distinct metabolic pathways, P2 receptor-mediated purinergic signaling, and monocarboxylate transporters 1 and 2 (MCT1, MCT2) in E. bovis sporozoite-induced NET formation. Seahorse-based experiments revealed a rapid induction of both neutrophil oxygen consumption rate (OCR) and early glycolytic responses, thereby reflecting immediate PMN activation and metabolic changes upon confrontation with sporozoites. The impact of these metabolic changes on NET formation was studied via chemical inhibition experiments targeting glycolysis and energy generation by the use of 2-fluor-2-deoxy-D-glucose (FDG), 6-diazo-5-oxo-L-norleucin (DON), sodium dichloroacetate (DCA), oxythiamine (OT), sodium oxamate (OXA), and oligomycin A (OmA) to block glycolysis, glutaminolysis, pyruvate dehydrogenase kinase, pyruvate dehydrogenase, lactate dehydrogenase, and mitochondrial ATP-synthase, respectively. Overall, sporozoite-induced NET formation was significantly diminished via PMN pretreatments with OmA and OXA, thereby indicating a key role of ATP- and lactate-mediated metabolic pathways. Consequently, we additionally studied the effects of extracellular pH, MCT1, MCT2, and purinergic receptor inhibitors (AR-C141900, AR-C155858, theobromine, and NF449, respectively). Pretreatment with the latter inhibitors led to blockage of sporozoite-triggered DNA release from exposed bovine PMN. This report provides first evidence on the pivotal role of carbohydrate-related metabolic pathways and purinergic receptors being involved in E. bovis sporozoite-induced NETosis.

d-lactate-triggered extracellular trap formation in cattle polymorphonuclear leucocytes is glucose metabolism dependent.

D-lactic acidosis is a metabolic disease of cattle caused by the digestive overgrowth of bacteria that are highly producers of d-lactate, a metabolite that then reaches and accumulates in the bloodstream. d-lactate is a proinflammatory agent in cattle that induces the formation of extracellular traps (ETs) in polymorphonuclear leucocytes (PMN), although information on PMN metabolic requirements for this response mechanism is insufficient. In the present study, metabolic pathways involved in ET formation induced by d-lactate were studied. We show that d-lactate but not l-lactate induced ET formation in cattle PMN. We analyzed the metabolomic changes induced by d-lactate in bovine PMN using gas chromatography-mass spectrometry (GC-MS). Several metabolic pathways were altered, including glycolysis/gluconeogenesis, amino sugar and nucleotide sugar metabolism, galactose metabolism, starch and sucrose metabolism, fructose and mannose metabolism, and pentose phosphate pathway. d-lactate increased intracellular levels of glucose and glucose-6-phosphate, and increased uptake of the fluorescent glucose analog 2-NBDG, suggesting improved glycolytic activity. In addition, using an enzymatic assay and transmission electron microscopy (TEM), we observed that d-lactate was able to decrease intracellular glycogen levels and the presence of glycogen granules. Relatedly, d-lactate increased the expression of enzymes of glycolysis, gluconeogenesis and glycogen metabolism. In addition, 2DG (a hexokinase inhibitor), 3PO (a 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 inhibitor), MB05032 (inhibitor of fructose-1,6-bisphosphatase) and CP-91149 (inhibitor of glycogen phosphorylase) reduced d-lactate-triggered ETosis. Taken together, these results suggest that d-lactate induces a metabolic rewiring that increases glycolysis, gluconeogenesis and glycogenolysis, all of which are required for d-lactate-induced ET release in cattle PMN.